This invention relates to protective Helicobacter antigens, especially H. pylori antigens, and in particular to the use of these antigens for the treatment of, or prevention of, gastroduodenal disease associated with H. pylori infection.
Helicobacter pylori is a gram negative, spiral bacterium which infects the lining of the human stomach. It is widely distributed, chronically infecting perhaps half the world""s population. The bacterium spreads from person to person by oral-oral or faecal-oral transmission, there being no recognised environmental reservoir.
Infection with the bacterium causes an inflammation of the gastric mucosa, or stomach lining. Usually this does not resolve, and infection and inflammation are believed to persist for many decades. Often this is not associated with symptoms, however this chronic infection is associated with an increased risk of a number of sequelae. A significant portion of those infected develop peptic ulceration of the duodenum or stomach, when the infection process disrupts the usual protective mechanisms the stomach has against its own digestive products. Also, long periods of infection increase the risk of the development of adenocarcinomas or lymphomas of the stomach wall.
Therefore, prevention or treatment of H. pylori infection has the potential to prevent considerable mortality and morbidity resulting from the sequelae of chronic infection.
In early experiments, H. pylori did not infect conventional laboratory animals. However, a laboratory mouse model of H. pylori infection, using the closely related organism, Helicobacter felis, has been developed (Lee et al., 1990; Dick-Hegedus and Lee, 1991). This model has proven very useful in screening new antimicrobial therapeutic regimes.
H. felis is a spiral shaped bacterium that shares a very close DNA homology with H. pylori. The bacterium colonises the mouse stomach in a similar manner to the way that H. pylori colonises the human stomach. The main ecological niche is gastric mucus, and colonisation is mainly seen in the antrum of the stomach. In germfree mice, H. felis infection induces a gastritis that is very similar to the human H. pylori infection, with a chronic inflammation of mononuclear cells accompanied by a polymorphonuclear leucocyte infiltration. Infection with either organism results in the induction of a similar raised systemic humoral immune response against H. pylori and H. felis respectively (Lee et al., 1990).
The H. felis model has proved to be very predictive of the efficacy of anti-H. pylori therapy in humans. Thus, monotherapy with agents with high in vitro activity such as erythromycin show no significant in vivo effect against H. felis in mice, just as erythromycin has no ant-H. pylori effect in humans, despite its high antimicrobial effects in vitro. In contrast, the triple therapy regimens of a bismuth compound, metronidazole, and tetracycline or amoxycillin lead to a very high eradication rate in H. felis infected mice (Dick-Hegedus and Lee, 1991). Such therapies are among the most successful human anti-H. pylori regimens.
The H. felis model has also been used to demonstrate that mice can be orally immunised with Helicobacter antigens, either to protect them from becoming infected (Chen et al, 1992), or to treat them when they are already infected so as to eradicate the infection (Doidge et al, 1994). Antigens that have been used in these vaccines include disrupted cellular preparations from either H. felis or H. pylori, and the bacterial enzyme urease from H. felis or H. pylori or subunits thereof, produced from E. coli clones expressing all or part of the H. pylori urease molecule (Michetti et al, 1994; see also International Patent Publications Nos. WO 90/04030, WO 93/07273 and WO 94/09823). H. pylori heat shock protein (Hsp or HSP) has also been shown to be a protective antigen (Ferrero et al., 1995).
International Patent Publication No. WO 93/18150 (Application No. PCT/EP93/00472) discloses vaccines or therapeutic compositions comprising one or more of recombinant H. pylori cytotoxin (CT or VacA), H. pylori cytotoxin-associated immunodominant antigen (CAI or CagA) or H. pylori heat shock protein, optionally together with H. pylori urease. International Patent Publication No. WO 95/27506 (Application No. PCT/FR95/00383) discloses an anti-H. pylori immunising composition containing a substantially purified H. pylori catalase as the active ingredient; and International Patent Publication No. WO 95/14093 (Application No. PCT/EP93/03259) discloses an immunogenic composition capable of inducing protective antibodies against Helicobacter infection which comprises at least one urease structural polypeptide from H. pylori or H. felis and optionally a urease-associated heat shock protein or chaperonin from Helicobacter.
The fact that antigens derived from H. pylori can be used to protect mice from H. felis infection suggests that there are cross-reactive, and cross-protective antigens between the two species. That is, that there are molecules present in H. pylori, which can induce immune responses in mice that recognise targets on H. felis, thus protecting the mice from H. felis infection. If an immune response to these H. pylori molecules will protect mice from H. felis infection, it is likely that similar immune responses will protect humans from H. pylori infection, or if already infected, cure them of it. Urease has been demonstrated to be such a cross-protective molecule in the H. felis mouse model (Michetti et al, 1994).
In work leading to the present invention, in order to identify further cross-reactive and cross protective antigens, a DNA library from an H. pylori strain has been constructed and screened with serum from mice that had been orally immunised with a vaccine prepared from disrupted H. felis cells and a mucosal adjuvant, with the aim of identifying E. coli clones expressing H. pylori proteins recognised by anti-H. felis antibodies and of subsequently identifying the antigenic protective H. pylori proteins.
In one aspect, the present invention provides an antigenic preparation for use in the treatment or prevention of Helicobacter infection in a mammalian host, which comprises an at least partially purified preparation of at least one Helicobacter antigen selected from the group consisting of:
(i) an antigen having a molecular mass of approximately 19 kDa which is processed into a mature form having a molecular mass of approximately 17 kDa;
(ii) an antigen having a molecular mass of approximately 13 kDa;
(iii) an antigen having a molecular mass of approximately 36 kDa;
(iv) an antigen having a molecular mass of approximately 50 kDa;
(v) an antigen having a molecular mass of approximately 29 kDa; and
(vi) immunogenic fragments of any of antigens (i) to (v) above which are capable of eliciting a specific protective immune response in a mammalian host.
In another aspect, the present invention provides an isolated Helicobacter antigen for use in the treatment or prevention of Helicobacter infection in a mammalian host, selected from the group consisting of:
(i) an antigen having a molecular mass of approximately 19 kDa which is processed into a mature form having a molecular mass of approximately 17 kDa;
(ii) an antigen having a molecular mass of approximately 13 kDa;
(iii) an antigen having a molecular mass of approximately 36 kDa;
(iv) an antigen having a molecular mass of approximately 50 kDa; and
(v) an antigen having a molecular mass of approximately 29 kDa; and
(vi) immunogenic fragments of any of antigens (i) to (v) above which are capable of eliciting a specific protective immune response in a mammalian host.
Each of the above antigens is further characterised by being reactive with anti-H. felis antibodies.
Preferably, antigen (i) above comprises an amino acid sequence substantially corresponding to the deduced sequence of clone B4.6 hereinafter (SEQ ID NO.10), or allelic or other variants thereof; antigen (ii) above comprises an amino acid sequence substantially corresponding to the deduced sequence of clone C3.5 hereinafter (SEQ ID NO.2), or allelic or other variants thereof; antigen (iii) above comprises an amino acid sequence substantially corresponding to the deduced sequence of clone E2.5 hereinafter (SEQ ID NO.4), or allelic or other variants thereof; antigen (iv) above comprises an amino acid sequence substantially corresponding to the deduced sequence of clone G3.8 hereinafter (SEQ ID NO. 6), or allelic or other variants thereof; and antigen (v) above comprises an amino acid sequence substantially corresponding to the deduced sequence of clone H5.1 hereinafter (SEQ ID NO. 8), or allelic or other variants thereof.
Suitable variants may have at least 50-60%, more preferably at least 70-80%, and most preferably at least 90%, similarity to one of the amino acid sequences referred to above, or to a region or part thereof, provided the variant is capable of eliciting a specific protective immune response in a mammalian host.
The term xe2x80x9cat least partially purifiedxe2x80x9d as used herein denotes a preparation in which the content of the particular antigen is greater, preferably at least 30% greater and more preferably at least 50% greater, than the content of the antigen in a whole cell sonicate of Helicobacter bacteria. Preferably, the preparation is one in which the antigen is xe2x80x9csubstantially purexe2x80x9d, that is one in which the content of the particular antigen is at least 80%, more preferably at least 90%, of the total Helicobacter antigens in the preparation.
The term xe2x80x9cisolatedxe2x80x9d as used herein denotes that the antigen has undergone at least one purification or isolation step, and preferably the antigen is in a form suitable for use in a vaccine composition.
It is to be understood that the present invention extends not only to the particular antigens of Helicobacter bacteria as described above, but also to immunogenic fragments of the particular antigen(s), that is fragments of the antigen(s) which are capable of eliciting a specific protective immune response in a mammalian host. Suitably, the immunogenic fragment will comprise at least five, and more preferably at least ten, contiguous amino acid residues of the particular antigen(s). Such immunogenic fragments may also be recognised by Helicobacter-specific antibodies, particularly antibodies which have a protective or therapeutic effect in relation to Helicobacter infection.
In another aspect, the present invention provides a vaccine composition for use in the treatment or prevention of Helicobacter infection in a mammalian host, which comprises an immunologically effective amount of an antigenic preparation or of an isolated Helicobacter antigen as broadly described above, optionally in association with an adjuvant, together with one or more pharmaceutically acceptable carriers and/or diluents.
In yet another aspect, the present invention provides a method for the treatment or prevention of Helicobacter infection in a mammalian host, which comprises administration to said host of an immunologically effective amount of an antigenic preparation or of an isolated Helicobacter antigen as broadly described above, optionally in association with an adjuvant.
In a related aspect, this invention provides the use of a vaccine composition comprising an immunologically effective amount of an antigenic preparation or of an isolated Helicobacter antigen as broadly described above, optionally in association with an adjuvant, for the treatment or prevention of Helicobacter infection in a mammalian host.
By use of the term xe2x80x9cimmunologically effective amountxe2x80x9d herein, it is meant that the administration of that amount to a mammalian host, either in a single dose or as part of a series, is effective for treatment or prevention of Helicobacter infection. This amount varies depending upon the health and physical condition of the individual to be treated, the taxonomic group of individual to be treated, the capacity of the individual""s immune system to synthesise antibodies, the degree of protection desired, the formulation of the vaccine, the assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials.
Preferably, but not essentially, the antigenic preparation of this invention is orally administered to the host, and is administered in association with a mucosal adjuvant. However, the invention also extends to parenteral administration of this antigenic preparation.
The present invention also extends to an antibody, which may be either a monoclonal or polyclonal antibody, specific for an antigenic preparation or an isolated Helicobacter antigen as broadly described above. Such antibodies may be produced by methods which are well known to persons skilled in this field.
In this aspect, the invention further provides a method for the treatment or prevention of Helicobacter infection in a mammalian host, which comprises passive immunisation of said host by administration of an effective amount of an antibody, particularly a monoclonal antibody, specific for an antigenic preparation or an isolated Helicobacter antigen as broadly described above.
The Helicobacter antigenic preparation or isolated antigen of this invention may be prepared by purification or isolation from natural sources, such as a whole cell sonicate of Helicobacter bacteria. Alternatively, however the antigenic preparation or isolated antigen may be prepared by synthetic, preferably recombinant, techniques. When prepared by recombinant techniques, the antigen may have an amino acid sequence substantially identical to the naturally occurring sequence or may contain one or more amino acid substitutions, deletions and/or additions thereto provided that following such alterations to the sequence, the molecule is still capable of eliciting a specific protective immune response against the naturally occurring Helicobacter antigen. A similar immunogenic requirement is necessary for any fragments or derivatives of the antigen whether made from the recombinant molecule or the naturally occurring molecule. Accordingly, reference herein to a Helicobacter antigen is considered reference to the naturally occurring molecule, its recombinant form and any mutants, derivatives, fragments, homologues or analogues thereof provided that such molecules elicit a specific protective immune response against the naturally occurring Helicobacter antigen. Also included are fusion molecules between two or more Helicobacter antigens or with other molecules including fusion molecules with other molecules such as glutathione-S-transferase (GST) or xcex2-galactosidase.
The present invention also extends to an isolated nucleic acid molecule encoding a Helicobacter antigen of the present invention, and preferably having a nucleotide sequence as set forth in one of SEQ ID NO. 1, 3, 5, 7 or 9, or being substantially similar to all or a part thereof. The term xe2x80x9csubstantially similarxe2x80x9d means having at least 40-50%, more preferably at least 60-70%, and most preferably at least 80% identity. A xe2x80x9cpartxe2x80x9d in this context means a contiguous series of at least 15 nucleotides, and more preferably at least 25 nucleotides.
According to this embodiment, there is provided a nucleic acid molecule comprising a sequence of nucleotides which encodes a Helicobacter antigen as broadly described above, and hybridises under low stringency conditions to all or part of a nucleic acid sequence set forth in one of SEQ ID NO. 1, 3, 5, 7 or 9, or to a complementary form thereof.
In another aspect, this invention provides a nucleic acid molecule comprising a sequence of nucleotides substantially as set forth in one of SEQ ID NO. 1, 3, 5, 7 or 9, or a part thereof.
The nucleic acid molecule may be RNA or DNA, single stranded or double stranded, in linear or covalently closed circular form. For the purposes of defining the level of stringency, reference can conveniently be made to Manratis, et al. (1982) which is herein incorporated by reference where the washing step at paragraph 11 is considered high stringency. A low stringency is defined herein as being in 0.1-0.5 w/v SDS at 37-45xc2x0 C. for 2-3 hours. Depending on the source and concentration of nucleic acid involved in the hybridisation, alternative conditions of stringency may be employed such as medium stringent conditions which are considered herein to be 0.25-0.5% w/v SDS at xc2x145xc2x0 C. for 2-3 hours or high stringent conditions as disclosed by Maniatis, et al. (1982).
It will be appreciated that the sequence of nucleotides of this aspect of the invention may be obtained from natural, synthetic or semi-synthetic sources; furthermore, this nucleotide sequence may be a naturally-occurring sequence, or it may be related by mutation, including single or multiple base substitutions, deletions, insertions and inversions, to such a naturally-occurring sequence, provided always that the nucleic acid molecule comprising such a sequence is capable of being expressed as a Helicobacter antigen as broadly described above.
The nucleotide sequence may have expression control sequences positioned adjacent to it, such control sequences usually being derived from a heterologous source.
This invention also provides a recombinant DNA molecule comprising an expression control sequence having promoter sequences and initiator sequences and a nucleotide sequence which codes for a Helicobacter antigen, the nucleotide sequence being located 3xe2x80x2 to the promoter and initiator sequences. In yet another aspect, the invention provides a recombinant DNA cloning vehicle capable of expressing a Helicobacter antigen comprising an expression control sequence having promoter sequences and initiator sequences, and a nucleotide sequence which codes for a Helicobacter antigen, the nucleotide sequence being located 3xe2x80x2 to the promoter and initiator sequences. In a further aspect, there is provided a host cell containing a recombinant DNA cloning vehicle and/or a recombinant DNA molecule as described above.
Suitable expression control sequences and host cell/cloning vehicle combinations are well known in the art, and are described by way of example, in Sambrook et al. (1989).
In yet further aspects, there is provided fused polypeptides comprising a Helicobacter antigen of this invention and an additional polypeptide, for example a polypeptide coded for by the DNA of a cloning vehicle, fused thereto. Such a fused polypeptide can be produced by a host cell transformed or infected with a recombinant DNA cloning vehicle as described above, and it can be subsequently isolated from the host cell to provide the fused polypeptide substantially free of other host cell proteins.
The present invention also extends to synthetic polypeptides displaying the antigenicity of a Helicobacter antigen of this invention. As used herein, the term xe2x80x9csyntheticxe2x80x9d means that the polypeptides have been produced by chemical or biological means, such as by means of chemical synthesis or by recombinant DNA techniques leading to biological synthesis. Such polypeptides can, of course, be obtained by cleavage of a fused polypeptide as described above and separation of the desired polypeptide from the additional polypeptide coded for by the DNA of the cloning vehicle by methods well known in the art. Alternatively, once the amino acid sequence of the desired polypeptide has been established, for example, by determination of the nucleotide sequence coding for the desired polypeptide, the polypeptide may be produced synthetically, for example by the well-known Merrifield solid-phase synthesis procedure.
Once recombinant DNA cloning vehicles and/or host cells expressing a Helicobacter antigen of this invention have been identified, the expressed polypeptides synthesised by the host cells, for example, as a fusion protein, can be isolated substantially free of contaminating host cell components by techniques well known to those skilled in the art.
Isolated polypeptides comprising, or containing in part, amino acid sequences corresponding to a Helicobacter antigen may be used to raise polyclonal antisera by immunising rabbits, mice or other animals using well established procedures. Alternatively, such polypeptides may be used in the preparation of monoclonal antibodies by techniques well known in the art.
In addition, the polypeptides in accordance with this invention including fused polypeptides may be used as an active immunogen in the preparation of single or multivalent vaccines by methods well known in the art of vaccine manufacture for use in the treatment or prevention of Helicobacter infection in a mammalian host.
Alternatively, the polypeptides in accordance with the present invention including fused polypeptides may be used as antigen in a diagnostic immunoassay for detection of antibodies to Helicobacter in a sample, for example, a serum sample from a human or other mammalian patient. Such immunoassays are well known in the art, and include assays such as radioimmunoassays (RIA) and enzyme-linked immunosorbent assays (ELISA).
Throughout this specification, unless the context requires otherwise, the word xe2x80x9ccomprisexe2x80x9d, or variations such as xe2x80x9ccomprisesxe2x80x9d or xe2x80x9ccomprisingxe2x80x9d, is to be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.
Preferably, the antigenic preparation or isolated antigen of this invention comprises H. pylori or H. felis antigen(s). Preferably also, this antigenic preparation or isolated antigen is used in a vaccine composition for oral administration which includes a mucosal adjuvant.
In a particularly preferred aspect of this invention, an oral vaccine composition comprising an antigenic preparation or isolated antigen comprising H. pylori antigen(s) as broadly described above, in association with a mucosal adjuvant, is used for the treatment or prevention of H. pylori infection in a human host.
The mucosal adjuvant which is optionally, and preferably, administered to the infected host with the Helicobacter antigenic preparation of this invention, is preferably cholera toxin. Mucosal adjuvants other than cholera toxin which may be used in accordance with the present invention include non-toxic derivatives of cholera toxin, such as the B sub-unit (CTB), chemically modified cholera toxin, or related proteins produced by modification of the cholera toxin amino acid sequence. These may be added to, or conjugated with, the Helicobacter antigenic preparation. The same techniques can be applied to other molecules with mucosal adjuvant or delivery properties such as Escherichia coli heat labile toxin. Other compounds with mucosal adjuvant or delivery activity may be used such as bile; polycations such as DEAE-dextran and polyornithine; detergents such as sodium dodecyl benzene sulphate; lipid-conjugated materials; antibiotics such as streptomycin; vitamin A; and other compounds that alter the structural or functional integrity of mucosal surfaces. Other mucosally active compounds include derivatives of microbial structures such as MDP; acridine and cimetidine.
The Helicobacter antigenic preparation or isolated antigen of this invention may be delivered in accordance with this invention in ISCOMS(trademark) (immune stimulating complexes), ISCOMS(trademark) containing CTB, liposomes or encapsulated in compounds such as acrylates or poly(DL-lactide-co-glycoside) to form microspheres of a size suited to adsorption by M cells. Alternatively, micro or nanoparticles may be covalently attached to molecules such as vitamin B12 which have specific gut receptors. The Helicobacter antigenic preparation or isolated antigen may also be incorporated into oily emulsions and delivered orally. An extensive though not exhaustive list of adjuvants can be found in Cox and Coulter, (1992).
Other adjuvants, as well as conventional pharmaceutically acceptable carriers, excipients, buffers or diluents, may also be included in the prophylactic or therapeutic vaccine composition of this invention. The vaccine composition may, for example, be formulated in enteric coated gelatine capsules including sodium bicarbonate buffers together with the Helicobacter antigenic preparation or isolated antigen and cholera toxin mucosal adjuvant.
The formulation of such prophylactic or therapeutic vaccine compositions is well known to persons skilled in this field. Suitable pharmaceutically acceptable carriers and/or diluents include any and all conventional solvents, dispersion media, fillers, solid carriers, aqueous solutions, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art, and it is described, by way of example, in Remington""s Pharmaceutical Sciences, 18th Edition, Mack Publishing Company, Pennsylvania, USA. Except insofar as any conventional media or agent is incompatible with the active ingredient, use thereof in the vaccine compositions of the present invention is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
The Helicobacter antigenic preparation or isolated antigen of the present invention may be administered as the sole active immunogen in a vaccine composition. Alternatively, however, the vaccine composition may include other active immunogens, including other Helicobacter antigens such as urease, lipopolysaccharide, Hsp60, VacA, CagA or catalase, as well as immunologically active antigens against other pathogenic species.
As an alternative to the delivery of the Helicobacter antigenic preparation or isolated antigen in the form of a therapeutic or prophylactic vaccine composition, the antigen or an immunogenic fragment thereof may be delivered to the mammalian host using a live vaccine vector, in particular using live recombinant bacteria, viruses or other live agents, containing the genetic material necessary for the expression of the antigen of immunogenic fragment as a foreign polypeptide. Particularly, bacteria that colonise the gastrointestinal tract, such as Salmonella, Shigella, Yersinia, Vibrio, Escherichia and BCG have been developed as vaccine vectors, and these and other examples are discussed by Holmgren et al. (1992) and McGhee et al.(1992).
Accordingly, the present invention also extends to delivery to the host using a vaccine vector expressing an isolated Helicobacter antigen as broadly described above, or an immunogenic fragment thereof. Accordingly, in a further aspect this invention provides a preparation for use in the treatment or prevention of Helicobacter infection in a mammalian host, which comprises a vaccine vector expressing an isolated Helicobacter antigen as broadly described above, or an immunogenic fragment thereof.
In this aspect, the invention extends to a method for the treatment or prevention of Helicobacter infection in a mammalian host, which comprises administration to said host of a vaccine vector expressing an isolated Helicobacter antigen as broadly described above or an immunogenic fragment thereof.
Further, the invention extends to the use of a vaccine vector expressing an isolated Helicobacter antigen as broadly described above, or an immunogenic fragment thereof, for the treatment or prevention of Helicobacter infection in a mammalian host.
Further features of the present invention are more fully described in the following Examples. It is to be understood, however, that this detailed description is included solely for the purposes of exemplifying the present invention, and should not be understood in any way as a restriction on the broad description of the invention as set out above.
FIGS. 1A and B, 2A and B show cloned H. pylori proteins expressed from E.coli XLOLR;
(1A) analysed on 4-20% gradient SDS-polyacrylamide gels, and visualised by CBB stain. Lane M, Molecular weight standards (kDa); Lane 1, Family A; Lane 2, Family B; Lane 3, Family C; Lane 4, uncharacterized protein; Lane 5, Family F; Lane 6, Family G; Lane 7, Family H; Lane 8, Negative Control, E.coli XLOLR; Lane 9, Positive Control, Helicobacter pylori total cell proteins;
(1B) corresponding Western blot samples, lane order the same as for panel A; analyzed on 4-20% gradient SDS-polyacrylamide gels, and visualized by CBB stain. Lane M, Molecular weight standards (kDa); Lanes 1 and 3, uncharacterized protein, Lane 2, Famile E; Lane between lanes 3 and 4, uncharacterized protein; lane 4, negative control.
(2B) corresponding Western Blot samples, lane order the same as for panel A.